Mason Lab

Problem : Biofilm formation, essential nutrient acquisition and resistance to mechanisms of host defense, all play key roles in nontypeable Haemophilus influenzae (NTHI) survival and ability to induce a disease state. There is a clear critical need to understand how NTHI regulates essential metabolic activities while countering immune defense strategies of the host.

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Actin polymerization coincides with NTHI adherence. Chinchilla nasopharyngeal epithelial (CNPE) cells were inoculated with 86-028NP sapA::kan containing a constitutively expressed GFP-reporter vector (Green) and incubated for four days. CNPE cells were labeled with Alexafluor-labeled ceramide (Red) and actin polymerization is observed by phaloidin labeling (Purple).

Wild type NTHI were cultured on primary chinchilla middle ear epithelial cells (CMEE) for four days and then analyzed for biofilm formation by optical sectioning microscopy. Bacteria contained a GFP-reporter vector that was constitutively expressed (green). CMEE cells were stained with Alexafluor-labeled ceramide (red).

We utilize a chinchilla animal model of NTHI-induced otitis media and live animal imaging to study bacterial virulence determinant expression in vivo. Here, a chinchilla was inoculated intranasally and transbullarly with an NTHI derivative strain that contains the lux genes under the control of the sap operon promoter. Sap operon expression is upregulated in vivo.

Background : Currently, it is unknown how commensal NTHI make the transition to that of opportunistic pathogen, thus promoting its survival in privileged host sites. NTHI is a gram negative obligate opportunistic pathogen of both the upper and lower respiratory tracts, responsible for disease of the middle ear, sinusitis, pneumonia, exacerbations in patients with chronic obstructive pulmonary disease and cystic fibrosis.

Targeting specific NTHI metabolic processes and mechanisms of resistance to the host innate immune response will promote development of therapies to improve the medical management of these diseases, thereby enhancing human health.

The Sap translocator, in complex with its substrate-binding protein, SapA, mediates uptake of heme and neutralization of host antimicrobial peptide lethality, both functions essential for NTHI survival as a commensal and opportunist. Sap function is both necessary and sufficient for disease development and thereby, a viable therapeutic target to alter NTHI-induced disease states. Sap translocator function is required for NTHI pathogenic behaviors that enable survival outside its commensal niche. Targeting this function for disruption will thus thwart the bacterium’s ability to cope with nutrient limitations and host defense mechanisms; functions required for disease development.

Significance : Commensal microorganisms must sense and adapt to various microenvironmental cues to establish long-term colonization in order to survive within the host. Mechanisms of nutrient acquisition, in concert with defense against host factors of immunity designed to protect against bacterial infection, stage a dynamic interplay between a commensal and its host niche. Disruption of this delicate balance can thwart commensal-host homeostasis and thus potentiate disease development. A better understanding of factors, both host and microbe related that support commensal behavior will help define bacterial targets that are exploited during opportunistic disease states. The Sap transporter mediates uptake of heme and neutralization of host antimicrobial peptides, functions essential for Haemophilus survival as both a commensal and opportunist. Our investigations will increase our understanding of how Haemophilus forms biofilms and interacts with host cells to achieve homeostatic state of commensalism. Our studies will allow development of small molecule inhibitors to block the essential functions of Sap as a mechanism to thwart infectious disease states and thus significantly reduce the clinical and economic burden for the diagnosis and treatment of NTHI-mediated diseases.